In order to reveal the molecular regulatory mechanisms of flavonoids biosynthesis in barley(Hordeum vulgare), the full length cDNA encoding of cinnamate-4-hydroxylase gene(HvC4H) was cloned from leaves of Qingke(hulless barley) through the methods of RT-PCR combining homologous clone and RACE technologies(Genbank accession number: KF927086). The full length was 1951bp , open reading frame (ORF) was 1518bp encoding 505 amino acids. Isoelectric point was 9.01 and grand average of hydropathicity (GRAVY) was -0.170, it meant the HvC4H was hydrophilic and alkaline protein. Advanced structure analysis showed HvC4H gene including family CYP450 conserved domains and specific functional active sites. The expression of HvC4H gene was analyzed in different tissues(stem, leaf and kernel) during 5 periods in endosperm development by real-time-fluorescence quantitative PCR(RTF-qPCR), the results showed obvious differences and the expression in stem was predominant . The study could lay the molecular foundation to raise the content of flavonoid in barley by controlling the expression of HvC4H gene and it provided useful informations to improve the quality, resistibility and growth of barley.